PROJECT SUMMARY/ABSTRACT Although protein S-palmitoylation was discovered more than 30 years ago and palmitoylated proteins have been implicated in pathogenesis of several diseases including cancer, cardiovascular and immune disorders, it remains one of the most understudied post-translational protein modifications. High lability, one of the key features of protein palmitoylation, along with its prominent effect on the protein function makes it an attractive mechanism for the regulation of intracellular signaling. In particular, rapid changes in protein palmitoylation could provide a molecular basis for activation of the plasma membrane-localized signaling proteins by targeting them into specific plasma membrane subdomains. It has been established that activation of the T cell receptor (TCR) signaling pathway is critically dependent on palmitoylation of signaling proteins and their functional association with the liquid ordered plasma membrane subdomains (?lipid rafts?) in a palmitoylation-dependent manner. However, molecular mechanisms regulating T cell signaling protein palmitoylation and lipid raft partitioning remain largely unknown. In our preliminary experiments we found that physiologically relevant stimulation of the TCR complex leads to rapid increase in palmitoylation of the tyrosine kinase Lck which is detectable within minutes upon engagement of TCR. To interpret this finding, we propose a model in which the TCR complex recruits and activates plasma membrane associated palmitoyl acyltransferases (PATs) to increase palmitoylation and plasma membrane compartmentalization of T cell signaling proteins. We will investigate this hypothesis in two Specific Aims. In Aim 1 we will determine the role of rapid stimulus-dependent protein palmitoylation in activation of the TCR signaling pathway. We will analyze the dynamics and regulation of stimulus-dependent palmitoylation of Lck, LAT and other members of the TCR signaling pathway. In Aim 2 we will determine if plasma membrane-localized PATs mediate T cell activation. This Aim will potentially uncover a previously uncharacterized role of DHHC PATs in regulation of T cell signaling. Overall, we expect that implementation of a novel class of regulatory enzymes into the canonical TCR signaling pathway would greatly expand a range of potential therapeutic targets for diseases associated with altered T cell homeostasis.